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Ex vivo Re-vascularization in Porcine Liver Bioengineering - A Critical First Step Towards Effective Transplantation of Bioengineered Livers

Periodic Reporting for period 1 - Liver Bioengineering (Ex vivo Re-vascularization in Porcine Liver Bioengineering - A Critical First Step Towards Effective Transplantation of Bioengineered Livers)

Reporting period: 2015-08-01 to 2017-07-31

Liver transplantation is currently the only proven therapy to extend life of patients with terminal liver disease. Organ bioengineering and regenerative medicine are promising new technologies that can help reduce the burden of liver shortage by increasing the number of organs available for transplantation. However, current bioengineered livers lack a functional vascular network that can readily allow their transplantation into a living host. Previous studies by the candidate have shown successful decellularization of acellular liver scaffolds using endothelial cells and hepatic cells in a perfusion bioreactor, creating a vascularized human liver organoid. However, its vascular network was unable to maintain vascular patency under constant blood flow for long periods of time. Hence, better understanding of how experimental cell seeding conditions of porcine liver scaffolds influence their re-vascularization efficiency is critical to achieve sustainable vascular patency after transplantation. In order
to accomplish this, the impact of fluid flow pressure and the seeded cell number will be investigated in reendothelialization efficiency of an acellular porcine liver scaffold. Furthermore, bioreactor pre-conditioning with fluid flow pressure ramping and sequential cycles of vascular growth and maturation will be used to induce revascularization, maturation, and enhanced function to potentially increase vascular patency. Finally, revascularized liver scaffolds will be transplanted into 5-10Kg pigs and short and long-term vascular patency will be investigated. Hence, the long-term objective of this project is to create a functional re-vascularized porcine liver scaffold, a critical first step towards the effective transplantation of bioengineered livers.
Main Results
From all the objectives proposed initially, we were able to address almost all of them throughout the period of the project. However, we have to recognize that the proposed objectives were a bit over-ambitious, considering the available time. In this regard, a third year would have been instrumental in concluding the outstanding work. Nevertheless, the completed goals are proving instrumental in the ongoing lab research.

One of the main points of this project was the evaluation of the revascularized liver scaffolds after transplantation into pigs. During the development of the animal model, we had several technical issues that led to high mortality and were only solved recently, in the past few months. Hence, only now we can proceed with the transplantation of these liver scaffolds and understand the effect of our in vitro manipulations on the outcome in vivo.

On a general note, I believe that the proposed goal of understanding the effects of flow pressure and cell number on scaffold revascularization was achieved with a great contribution to the efficiency which we generate revascularized organ scaffolds. Similarly, we now understand some of the mechanisms necessary to generate functional vessels in these matrices, by modulating cycles of cell growth and maturation. This might not be enough to completely fulfill our goal of generating transplantable liver scaffolds, but it is certainly a huge step towards it, since we are now capable of generating complex vascular networks in vitro.
The World Health Organization estimates that some form of liver disease affects over 650 million people worldwide. Of those, over 21 million people are estimated to live with chronic liver disease, and about 800,000 expire annually. Despite the relatively slow progression of the disease, liver transplantation remains the only definitive treatment for end-stage chronic liver failure, and it is also the only treatment for severe acute liver failure and some forms of inborn errors of metabolism. Because organ donation has not kept up with demand in the past 15 years, the number of patients on the liver transplant waiting list has increased significantly. To overcome the shortage of livers for transplantation, several approaches have been developed such as artificial/bioartificial liver assist devices, new surgical methods (split liver method, living donation) and cellular therapies. Despite increasing the pool of available organs for transplantation, or extending the life of the patients on the liver waiting list for a short period of time, these approaches invariably fail to provide a long-term solution to organ shortage. Hence, new promising technologies like regenerative medicine, and more specifically organ bioengineering, will hopefully help to bridge the gap between the pool of available livers for transplantation and patients waiting for one. The candidate and others have recently developed methods for organ decellularization using detergent perfusion through the vasculature of solid organs. However, to successfully transplant a bioengineered liver it is critical that the newly bioengineered liver’s vascular network is fully functional and re-vascularized, able to maintain vascular patency and hemostasis once anastomosed with the host´s circulatory system. It is precisely here, that the current knowledge and technology is still quite limited making transplants of bioengineered organs unviable past a few hours, or at best a few days, because of blood clotting due to poor re-vascularization. Hence, it is imperative that more efficient revascularization methods can be investigated to improve bioengineered organ´s vascular patency and the probability of successful transplantation. In order to accomplish this, effective cell seeding is vital, followed by a gradual maturation phase of the newly developed and re-vascularized vascular structures, similarly to what has been proposed and implemented in tissue-engineered blood vessels.

We found that “near-physiological” higher fluid flow pressures in the portal vein (10-20mmHg) after cell seeding resulted in increased tissue growth and cell proliferation, while higher fluid flow pressures >50mmHg lead to cell death. Finally, we also found that within this range (10-20mmHg), there was a significant increase in the number of reendothelialized vascular structures, which was fluid flow pressure dependent, suggesting that precise mechanical stimulation is essential for efficient re-vascularization of acellular liver scaffolds ex vivo (Baptista et al. Tissue Engineering C - Methods, 2016). These results are in agreement with observations of small-for-size liver transplantations, where many have found that extreme hyperperfusion, indicative of high shear stress, leads to cell death and numerous other complications. Yet, mild elevations in flow rate are indispensible for liver regeneration following small-for-size transplantation, which we believe are correlated with fluid flow pressures within the 10-20mmHg range.

With this ultimate goal, we believe that this project will have a broad socio-economic impact, since it will help to generate some of the necessary technologies to create lab grown organs (with its own potential implications in intellectual property), adding more livers into the transplantation pool.